The present invention relates to a liquid-transfer device particularly useful as a capturing device for capturing substances in a liquid. The invention is especially useful in biological assay devices, wherein the substance in the liquid is a biological substance to be immobilized and captured for analysis purposes, or for reaction with another substance contained within another liquid.
The invention is described below with respect to devices and methods for performing accelerated analytic and synthetic analyses procedures, such as those relating immunological, genetic, biochemical and bioanalytical processes and biochemical assays. Such assays may be performed for a variety of purposes including, but not limited to, isolation and/or detection of proteins or polynucleotides, detection of blood group antigens and their antibodies, screening of drug candidate and compounds, life science research and clinical and molecular diagnostics.
Recent developments in a variety of research and diagnostic fields have created a need for improved and accelerated methods and apparatus for performing analytical, particularly, bioanalytical procedures and assays, to increase research efficiency and saving costs. Such need exists mainly in solid phase reactions which are time consuming processes.
Over the years, numerous biochemical processes were designed to be performed on different types of solid phase reactions including, biochemical processes, including synthesis, separation and extraction processes, diagnostic processes, and the like.
These biochemical processes were designed to be performed on different types of solid phase matrixes. Solid phase matrixes may by of non-water permeable material such as glass or polystyrene, which may be in form of tubes, microtitration plates, or microscopic slides. The solid phase may be comprised of a bibulous or porous membrane such as nylon or nitrocellulose or glass fibers and the like.
Biochemical reactions are driven by reactant collisions which are effected by energy kinetics of the reactants and their concentration. (The collision theory). “The physical basis of biochemistry: the foundations of molecular biophysics By Peter R. Bergethon, Edition: Published by Springer, 1998
In typical biochemical solid phase reactions such as immunoassay reactions, at least two types of reactants are involved. One type of reactant is immobilized to the solid phase (antibody or antigen), and other reactant, having binding affinity to the immobilized reactant, is free in the reaction solution. Reactant immobilization therefore reduces energy kinetics and collision frequency, and therefore prolongs reaction time. “ELISA and other solid phase immunoassays: theoretical and practical aspects By D. M. Kemeny, Stephen J. Challacombe, Edition: illustrated, Published by John Wiley and Sons, 1988”
In order to accelerate solid phase reactions, two type of solution were used. One type was a flow-through type of assay, in particular immunoassays type such as described in Valkirs et. al., U.S. Pat. No. 4,632,901. This patent disclose a device containing a porous binding membrane as a solid phase matrix to which receptor molecule are bound, and in which the binding membrane is in contact with an absorbent membrane. A heterogeneous reaction is effected in a short time, while a sample containing an analyte is applied on top of the binding membrane, followed by washing solutions and signal producing liquid.
A different type of flow through device was disclosed by Mabuchi et. al, U.S. Pat. Application Publication 2007/0243628, relating to a device for detection of proteins on a blotted membrane. The device comprises several layers including a support layer placed below the blotting membrane, and a flow distributor layer placed above the blotting membrane. The layers are held by a plastic housing having a reservoir for holding the reagents above the flow distributor.
The reaction liquids are transferred through the layers by vacuum or positive gas pressure. Such methods therein described are rapid, but flow control is difficult and the results will have low reproducibility.
A different approach to accelerate biochemical immunoassays is the use of lateral flow devices, sometimes also named as immuno-chromatography devices. Gordon et. al., U.S. Pat. No. 4,956,302 discloses such a device for the detection of antigens or antibodies in a fluid sample by lateral flow through a bibulous membrane strip enclosed in a plastic housing.
Another device is described by Bunce et. al. U.S. Pat. No. 5,198,193, which reduces the problem of formation of undesirable complexes produces by the close presence of samples and reagents in a dense porous material. The device comprises two liquid flow channels leading to a common site and operable to deliver liquid to this site in a sequentially timed manner following simultaneous application of such liquid to the channels. A capture zone is positioned in the common site or downstream of it.
Another dual path immunoassay device is disclosed by Esfandiari U.S. Pat. No. 7,189,522 intended to overcome interactions between sample and reaction reactants such as conjugates which lead to aggregation. The solution described in that patent was (a) to separate the flow of the sample from the flow of the conjugate by using two distinct flow paths, two inlets each in every path, and (b) to control the timing of the two flows so that at a particular time, only one flow is taking place in the capture zone. The device described is composed of two flow path perpendicular to each other. The two paths have portions which overlie one another and the capture zone is immobilized on one or both of the flow path material at the junction. The device is operated by applying the sample to one path, usually that having a higher pore size.
Such solutions do not solve the main problem in lateral flow, which is the contradiction between the requirement for rapid assay and high sensitivity. Rapid assay is achieved by fast lateral flow, due to high wicking property of the membrane, in Nitrocellulose membranes the wicking property is effected by pore size, Membranes of 5 micrometer have wicking rate of nearly 200 s/4 cm, as membranes of 15 micrometer have higher rate, around 70 s/4 cm. High sensitivity is obtained by high membrane binding capacity, lower pore size increase binding capacity, for example, in 0.1 μm Nitrocellulose membranes the protein binding capacity is 100-150 μg IgG/cm2 as for 0.45 μm membranes the binding capacity is 50-100 μg IgG/cm2.
Buechler et. al., U.S. Pat. No. 6,156,270, describes a system and a device for lateral flow immunoassay using a non bibulous, non porous, flow path. The device comprises two opposing surfaces disposed at a capillary distance apart. One of the sides has a capture zone in which receptor molecules are immobilized.
Eisinger et. al. U.S. Pat. No. 4,943,522 describes a lateral flow device having a non-bibulous membrane for conducting an immunoassay.
U.S. Pat. No. 5,202,268 teaches a multi-layered test card for the determination of substances in liquids wherein the liquids flow from a first membrane into a second membrane and back to the first membrane. In order to enable such unipath flow, each membrane is divided by liquid flow barriers in order to disattach the intimate contact between the membranes. This configuration prevents parallel flow and enables a sequential flow through the membranes.
A complex formed by a specific binding reaction is generally not directly observable. Various techniques have been devised for labeling one member of the specific binding pair to enable visualization and measurement of the complex. Known labels include radioactivity, gold particles, magnetic particles, chromophores, fluorophores and enzymes. When a member of a specific binding pair is conjugated to an enzyme, the complex may be detected by the enzymatic activation of a reaction system including a signal generating substrate/cofactor group wherein a compound, such as a dyestuff, is activated to produce a detectable signal.
One approach for increasing speed, and simplifying such binding assays performed on a solid phase matrix, utilizes a one step lateral flow capillary device such as depicted in FIG. 1, to be described below. Such choices are extremely useful as they are simple to operate even by an unskilled person, or under non-laboratory conditions, and they provide results in short duration.
The major drawback of one step lateral flow assays is their limited sensitivity. To obtain high assay sensitivity the assay should include several reaction steps including an enzymatic reaction step which amplifies the signal and preferably a washing step to reduce background and improve signal to noise ratio.
Performing multi-step assays in lateral flow devices may eliminate their major advantage which is short assay duration. Application and transport of several liquids serially through a typical capillary flow membrane substantially prolong the assay duration. The assay duration may be reduced by using high flow rate membranes, but such membranes have high pore size and relative low protein binding capacity, which results in low assay sensitivity. The efficiency of lateral flow based reactions is substantially affected by the receptor concentration immobilized in the capture zone.
From the above, it will be seen that it would be highly advantageous to have a lateral flow capillary device and method capable of performing rapid and simple reactions, particularly multi-step reactions, in the fields of biochemistry and medicine, particularly for research and diagnosis which avoid at least some of the above-discussed disadvantages of the prior art.